Bottom Line:
First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity.Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans.Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity.

fig6: Summary of the sources of enhanced affinity for glycoproteins binding to MGL. The KI values from Table 3 are linked by the fold enhancement resulting from various factors. Letters in parentheses refer to the illustrations in Fig. 1.

Mentions:
The glycoprotein presentation effect was investigated using the engineered glycoprotein ligands containing only a single glycosylation site as well as a glycopeptide pool from one of the variants. The results, illustrated in Fig. 5 and summarized in Table 3, reveal that the affinity of MGL for the three singly glycosylated variants of orosomucoid varies by a factor of less than 2, in spite of a range of nearly fivefold in the relative proportions of bi-and tri-antennary glycans attached to these variants. These results suggest that there is relatively little difference in the affinity of the receptor for the bi-and tri-antennary oligosaccharides. However, the relative affinity of the CRD of MGL for the glycopeptide from variant OR-1b (KI,Gal/KI,glycopeptide = 12.4; Table 3) is roughly fivefold higher than the 2.3-fold enhancement that would be expected based on the presence of an average of 2.3 terminal galactose residues per glycopeptide. This number is almost as high as the sevenfold enhancement observed for the tri-antennary glycopeptide from asialofetuin (Table 1), confirming that the degree of branching makes only a modest contribution to enhanced affinity. The most striking comparison is the difference in affinities for the intact glycoproteins bearing a single glycan (KI = 23 μM; Table 3) and the isolated glycopeptide derived from one of these glycoprotein (OR-1b, KI = 450 μM; Table 3), which reveals a 20-fold enhancement in affinity resulting from presentation of the glycoprotein rather than the glycopeptide. Thus, for a glycoprotein bearing a single glycan interacting with a single CRD, there is a 100-fold enhancement in affinity even when normalized to the number of terminal galactose residues present, resulting from the fivefold effect of the affinity of the CRD for branched glycans and the 20-fold effect of presentation of the glycan on a protein. These contributions are summarized in Fig. 6.

fig6: Summary of the sources of enhanced affinity for glycoproteins binding to MGL. The KI values from Table 3 are linked by the fold enhancement resulting from various factors. Letters in parentheses refer to the illustrations in Fig. 1.

Mentions:
The glycoprotein presentation effect was investigated using the engineered glycoprotein ligands containing only a single glycosylation site as well as a glycopeptide pool from one of the variants. The results, illustrated in Fig. 5 and summarized in Table 3, reveal that the affinity of MGL for the three singly glycosylated variants of orosomucoid varies by a factor of less than 2, in spite of a range of nearly fivefold in the relative proportions of bi-and tri-antennary glycans attached to these variants. These results suggest that there is relatively little difference in the affinity of the receptor for the bi-and tri-antennary oligosaccharides. However, the relative affinity of the CRD of MGL for the glycopeptide from variant OR-1b (KI,Gal/KI,glycopeptide = 12.4; Table 3) is roughly fivefold higher than the 2.3-fold enhancement that would be expected based on the presence of an average of 2.3 terminal galactose residues per glycopeptide. This number is almost as high as the sevenfold enhancement observed for the tri-antennary glycopeptide from asialofetuin (Table 1), confirming that the degree of branching makes only a modest contribution to enhanced affinity. The most striking comparison is the difference in affinities for the intact glycoproteins bearing a single glycan (KI = 23 μM; Table 3) and the isolated glycopeptide derived from one of these glycoprotein (OR-1b, KI = 450 μM; Table 3), which reveals a 20-fold enhancement in affinity resulting from presentation of the glycoprotein rather than the glycopeptide. Thus, for a glycoprotein bearing a single glycan interacting with a single CRD, there is a 100-fold enhancement in affinity even when normalized to the number of terminal galactose residues present, resulting from the fivefold effect of the affinity of the CRD for branched glycans and the 20-fold effect of presentation of the glycan on a protein. These contributions are summarized in Fig. 6.

Bottom Line:
First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity.Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans.Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity.